Estudio de la capacidad antiproliferante de xilopiranósidos a través de su actividad como aceptores trampa de la enzima beta (1,4) galactositransferasa 7. Expresión heteróloga de la enzima humana en escherichia coli

Resumen

Proteoglycans (PGs), including heparan sulfate (HS) forms, are important regulators of tumor progression. In the PGs biosynthetic process, the core protein is synthesized on a ribosomal template and the sugar chains are assembled posttranslationally, one sugar at a time, starting with the linkage of xylose to a serine residue of the core protein and followed by galactosydation of the xylosylprotein (Esko et al., 2009). This step is catalized by the xylosylprotein ß-1,4-galactosyltransferase 7 (B4GALT7) (Almeida et al., 1999; Okajima et al., 1999a). Hydrophobic xylopyranosides have been previously shown to prime glycosaminoglycan (GAG) synthesis, including GAG-HS forms, acting as a ¿decoy acceptors¿ of the B4GALT7 (Kolset et al., 1990). The compound 2-(6-hydroxynaphthyl) ß-D-xylopyranoside was show to cause growth inhibition of tumor cells, acting as a potent antiproliferative and anticarcinogen compound. This property was related to this ability to prime the HS synthesis (Mani et al., 1998; 2004). So, the ability of the xylosides to prime the assembly of HS chains ¿and hence their potential activity as antitumor agents¿ must be related to their ability to act as acceptors of B4GALT7. To know if the antiproliferative activity of synthetic xylopyranosides is related to their ability to act as "decoy acceptors" of B4GALT7, we have first heterologously expressed the catalytic domain of the human protein in Escherichia coli in a soluble Capítulo 1 4 and stable form as a fusion protein with a polihistidine tag. The recombinant protein was purified through homogenity an kinetically characterized. After that we have studied the ability of a variety of synthetic xylopyranoside derivatives to act as substrates or inhibitors of the recombinant enzyme. The xylopiranosides were synthesized as 3-amidopropyl derivatives, containing a common N-(O-xylopyranosyl)-hydoxylpropylamide moiety with a variable group that was selected to compare the effect of their diferent functional groups on the activity of the xyloside as decoy acceptor. The kinetic parameters for each single compound was obtained, showing a range of catalitic eficiencies that could be related to the structure of their aglycon moieties. Only the xylopyranosides with a voluminous aglicon groups show activity as decoy acceptors. To test if there is a relationship between the catalytic efficiency of the recombinant B4GALT7 with the different xylopyranosides and their antiproliferative activity, we assayed selected xylosides against the human lung carcinoma cell line A549. A representative sample of different xylosides was chosen attending to both their activities as decoy acceptors and their unique structural features. As expected, the xylosides that could not act as acceptor of B4GALT7 shows a lack of antiproliferative activity. The rest of xylopyranosides tested showed IC50 values lower than the reference compound 2-(6-hydroxynaphthyl) ß-D-xylopyranoside. A weak correlation between the activity as acceptors of B4GALT7 and activity as antiproliferatives of the xylopyranoside compounds was found. In conclusion, we have shown that it is possible to heterologously express the catalytic domain of the human B4GALT7, soluble and stable enough to undertake in vitro studies with it. This achievement opens the possibility of developing an easy-touse method to test the activity as decoy acceptors of natural and synthetic xylopyranosides. Since priming the synthesis of GAG is required but not enough for the antiproliferative activity of the xylosides, it is not possible to establish a direct strong correlation between the kinetic parameters of the recombinant B4GALT7 and the antiproliferative activity of the different xylopyranosides tested. On the other hand, preliminary results obtained in A549 cell line suggest that some xylopyranosides exhibit a promising antiproliferative activity.